Proteins of the TGF-β family function as cytokines mediating many important embryogenic and immune functions including chemotaxis, production of extracellular matrix, regulation of cell growth and differentiation, and development and regulation of the immune system. Thus, these molecules could be used in a great variety of therapies if available in sufficient quantities. Epithelial ovarian cancers, for example, are the fifth most common malignancy in women. Each year approximately 26,600 new cases of epithelial ovarian cancer are diagnosed, of which 55% die of the disease annually. Studies have shown that Müllerian Inhibiting Substance (MIS), a TGF-β family member, may potentially be an effective therapy for the highly lethal advanced ovarian carcinomas. The current production systems for MIS, including mammalian and bacterial systems, however, are not capable of providing MIS at levels required for clinical trials or commercial applications. Not only are these systems incapable of directly producing the biologically active C-terminal MIS, these systems cannot produce adequate quantities of the holo-MIS precursor and suffer from additional disadvantages as well.
Although the biotechnology industry has directed its efforts to eukaryotic hosts like mammalian cell tissue culture, yeast, fungi, insect cells, and transgenic animals, to express recombinant proteins, these hosts may suffer particular disadvantages. For example, although mammalian cells are capable of correctly folding and glycosylating bioactive proteins, the quality and extent of glycosylation can vary with different culture conditions among the same host cells. Yeast, alternatively, produces incorrectly glycosylated proteins that have excessive mannose residues, and generally exhibit limited post-translational processing. Other fungi may be available for high-volume, low-cost production, but they are not capable of expressing many target proteins. Although the baculovirus insect cell system can produce high levels of glycosylated proteins, these proteins are not secreted, however, thus making purification complex and expensive. Transgenic animal systems are hindered by lengthy lead times for developing herds with stable genetics, high operating costs, and potential contamination by prions or viruses.
Prokaryotic hosts such as E. coli may also suffer disadvantages in expressing heterologous proteins. For example, the post-translational modifications required for bioactivity may not be carried out in the prokaryote host. Some of these post-translational modifications include signal peptide processing, pro-peptide processing, protein folding, disulfide bond formation, glycosylation, gamma carboxylation, and beta-hydroxylation. As a result, complex proteins derived from prokaryote hosts are not always properly folded or processed to provide the desired degree of biological activity. Consequently, prokaryote hosts have generally been utilized for the expression of relatively simple foreign polypeptides that do not require post-translational processing to achieve a biologically active protein.
The biochemical, technical, and economic limitations on existing prokaryotic and eukaryotic expression systems has created substantial interest in developing new expression systems for the production of heterologous proteins. To that end, plants represent a suitable alternative to other host systems because of the advantageous economics of growing plant crops, plant suspension cells, and tissues such as callus; the ability to synthesize proteins in leaves, and storage organs like tubers, seeds, and fruits; the ability of plants to perform many of the post-translational modifications previously described, the capability of plants for protein bioproduction at very large scales; and the ability to produce the protein in an environment free of human pathogens. Plant-based expression systems may be more cost-effective than other large-scale expression systems for the production of therapeutic proteins.
The present invention contemplates producing a bioactive TGF-β protein, such as the MIS protein, in a plant host system. The MIS protein of the present invention may be any full length MIS precursor (e.g., holo-MIS), the 140 kD homodimer from which the bioactive C-terminal homodimer can be released, or the bioactive C-terminal peptide fragment which acts as a inhibitor of ovarian cancer at the desired concentration, and which under pathological conditions, modulates the functional activities of individual cells and tissues.
The MIS of the present invention belongs to the Transforming Growth Factor-beta (TGF-β) superfamily, which includes various TGF-β isoforms, GDF isoforms (Growth/Differentiation Factors), Inhibins, Activins, MIS (Müllerian Inhibiting Substance or Anti-Müllerian Hormone), BMP (bone morphogenetic proteins), dpp (decapentaplegic protein), Vg-1, and MNSF (monoclonal nonspecific suppressor factor). Proteins of this family share common features including sequence similarity, protein structure and post-translational processing, receptor interactions and biological function as cytokines. The MIS product of the present invention can act as a growth inhibitor of ovarian and other cancer cells originating in the reproductive organs of both males and females.
The MIS protein of the present invention has significant potential to serve as a novel, non-toxic and highly specific therapeutic agent for tumors of Müllerian origin. A wide variety of heterologous expression systems have been used in an attempt to increase yields of recombinant MIS protein. One system utilizes CHO cells transfected with the full length MIS coding sequence fused to the SV40 early promoter. Cate et al., 45 CELL 685-698 (1986). In this system, recombinant human MIS was secreted into the medium and recovered by immunoaffinity chromatography using a mouse monoclonal antibody to MIS. MacLaughlin et al., 131 ENDOCRINOLOGY 291-296 (1992); and Ragin et al., 3 PRO. EXPR. AND PUR. 236-245 (1992). Initial tests of the CHO-derived MIS showed that the majority of MIS was in a noncleaved (140 kD homodimer) form (Cate et al., 1986) which gave very limited antiproliferative activity in bioassays. Wallen et al., 49 CANC. RES. 2005-2011 (1989). However, additional steps involving MIS purification and proteolytic cleavage for C-terminal activation resulted in increased yields of bioactive protein. Kurian et al., 1 CLIN. CAN. RES. 343-349 (1995); and Ragin et al., 3 PRO. EXPR. AND PUR. 236-245 (1992). In vitro treatment with the protease plasmin has been used to generate complete cleavage and fully active MIS. In addition, enhanced cleavage efficiency was engineered into the MIS polypeptide by incorporating an arg-arg dibasic cleavage site in place of the native ser-arg site. Kurian et al., 1 CLIN. CAN. RES. 343-349 (1995). Stably transfected CHO lines which synthesize and secrete human holo-MIS have been developed. Kurian et al., 1995. However, scale-up to levels required for commercial production, or even for initial human clinical trials, has proven difficult and expensive using these systems. As a consequence, significant effort has been invested in analyzing alternative expression systems (E. coli, Pichia, baculovirus) for enhanced production of MIS. Although less costly, all of these systems were plagued with problems involving either production of insoluble aggregates or spurious proteolytic cleavage events and were less effective than the CHO-based expression system.
A number of additional observations support the need to search for more efficient means to produce MIS as an anticancer therapeutic. First, MIS activity is cleavage-dependent, and tumor cells in vitro do not have the ability to do this effectively. Thus, clinical applications may require administration of the fully activated form (C-terminal homo-dimer fragment). The apparent half-life of purified carboxyl-terminal MIS is short in vivo and thus, larger doses may be required. Attempts to express the C-terminal bioactive component directly in either bacterial or mammalian cell systems have been unsuccessful. It is not clear whether appropriate assembly of the C-terminal homo-dimer is dependent on the presence of the N-terminal pro-sequences or if its bioactivity in mammalian cells precludes effective bioproduction. The projected amounts of MIS required for initial in vitro and in vivo antiproliferative studies may be met by current protocols that involve CHO-based bioproduction of the holo-MIS and enzymatic activation in vitro. However, more effective strategies are needed to meet expected requirements for holo-MIS and carboxy-terminal MIS for later clinical trials and potential commercial markets. The method provided by the present invention will meet these needs by yielding more efficient and cost-effective means for producing bioactive therapeutic proteins that mimic the structure and biologically activity of authentic proteins.
Other objectives, features and advantages of the present invention will become apparent from the following detailed description. The detailed description and the specific examples, while indicating specific embodiments of the invention, are provided by way of illustration only. Accordingly, the present invention also includes those various changes and modifications within the spirit and scope of the invention that may become apparent to those skilled in the art from this detailed description.